Breast cancer is the most prevalent cancer among women worldwide [Parkin D M et al, CA Cancer 1− Clin. 55(2):74-108 (2005)]. To date conventional therapies for breast cancer such as surgery, chemotherapy and radiotherapy resulting in effective remission and diminishing risk of recurrence are still lacking. In the past few years accumulated data has indicated many solid tumors to be induced by cancer stem cells (CSCs) located within the tumor, denoted as “side population”. These side populations preserve their capacity to proliferate and undergo self renewal [Seo D C et al, Mol Cancer, 6(1):75 (2007)]. In breast cancer cell lines (2%) of the cells suspected as CSCs [Kopper and Hajdu, Pathol Oncol Res. 10(2):69-73 (2004)]. CSCs are difficult to treat due to the unique features they comprise. The CSCs contain an increased number of multi-drug resistant transporter proteins which pump out chemotherapeutic drugs from the cells. This characteristic is manifested by the pumping out of Hoechst 33342 and Rhodamine-123 dies from the cells. Also, the CSCs were shown to be more resistance to radiation [Woodward W A et al, 1− Cell Sci. 118 (Pt 16): 3585-94 (2005)]. Thus, understanding the molecular mechanisms governing CSCs fate decisions may allow for their divergence from proliferation to differentiation.
Two main signaling pathways have been suggested to play an essential role in inducing tumorigenicity in mammary cancer stem cells, the Wnt and the Notch signaling pathways. The Wnt signaling pathway has been suggested to play a role in oncogenesis of numerous types of cancers including colon, leukemia, melanoma and breast [Reya T and Clevers H Nature 434, 843-850, (2005), Brennan K R and Brown A M, J. Mammary Gland Biol. Neoplasia 9, 119-131 (2004)]. The Wnt signaling pathway is initiated by the binding of Wnt ligands to the Frizzled/LRP transmembrane receptors. As a result, the complex Axin/APC/GSK3beta which is involved in beta catenin degradation is inhibited resulting in beta catenin translocation and accumulation in the nucleus. Beta catenin together with the transcription factors LEF/TCF bind to the target genes promoters and induces transcription of genes such as cyclin D1 and c-myc which induce cell proliferation. In the absence of nuclear beta catenin, LEF/TCF, the transcription repressor Groucho and the histone deacetylases comprise a transcription repressive complex that acts to repress the expression of the Wnt target genes which may lead to cellular Differentiation.
The Wnt signaling pathway is modulated by the Dickkopf (Dkk) family proteins. Dkk family proteins consist of 4 members (Dkk1 to Dkk4). Dkk1, Dkk2 and Dkk4 have been previously suggested to play a predominantly inhibitory role in the regulation of the Wnt pathway and increase cell differentiation [Aguilera O et al, Carcinogenesis, 28(9):1877-84 (2007)]. However, recently accumulated data have indicated that Dkk2 may in fact activate the Wnt pathway [Niehrs C, Oncogene 25, 7469-7481 (2006)] and/or induce cell proliferation [Gregory C et al, J Biol Chem, 278(30):28067-78 (2003)]. In the canonical mechanism Dkk1 binds the LRP5/6 receptor at the plasma membrane, induces its internalization and thus prevents the binding of Wnt to the receptor [Niehrs C, Oncogene, 25, 7469-7481 (2006)], which results in Wnt signaling pathway inhibition.
In the recent years, large body of evidence has indicated Dkk family genes as tumor suppressor genes. Dkk are frequently silenced due to promoter hypermethylation in cancer cell lines such as gastrointestinal and other tumors [Sato H, Carcinogenesis, 28(12):2459-66 (2007)]. Moreover, ectopic expression of Dkk in various cancer cell lines resulted in sensitization to apoptosis [Mikheev A M et al Biochem Biophys Res Commun. 352(3):675-80 (2007), Mikheev A M et al Carcinogenesis 25(1):47-59 (2004)]. Ectopic expression of Dkk was also shown to reduced tumorigenity of cancer cell lines [Mikheev A M, et al, Carcinogenesis 25(1):47-59 (2004)].
Another major signaling pathway involves in cancer stem cell is the Notch pathway. Following the binding of Delta, Serrate, Lag-2 (DSL) ligand to Notch receptor, Notch receptor is undergone a series of proteolitic cleavages by several metalloproteases such as, A Disintegrin And Metalloproteinase (ADAM), ADAM 10 and ADAM 17, resulting in the release of Notch intracellular domain (NICD). NICD fragment translocates to the nucleus, where it up-regulates gene expression such as Hes and Hey [Wu F et al, Front Biosci, 12:4370-83 (2007)]. Notch signaling pathway can be inhibited by Numb which binds the intracellular domain of Notch receptor leading to its recruitment into endocytic vesicles [Berdnik D et al, Dev. Cell, 3 (2):221-31 (2002)].
The Notch and the Wnt signaling pathways are cross-talk. In the promoter region of Numb two TCF/LEF binding sites have discovered and thus it was suggested to be a target for the Wnt pathway [Katoh M and Katoh M, Int J Mol Med. 18(3):517-21 (2006)]. In addition, activation of LEF/TCF up-regulate the Notch ligand Jag 1 (Katoh M and Katoh M Int J Mol Med, 17(4):681-5 2006).
Another important aspect concerning cancer stem cells is the effect of cell-cell contact formed by the adhesion molecule E-cadherin. Changes in E-cadherins expression may indicate cell condition. During cell differentiation the expression of E-cadherins is enhanced whereas deregulation of E-cadherins has been linked to lose of tissue organization and carcinogenesis. E-cadherin influences other signaling pathways such as the Wnt pathway: E-cadherin is connected to the cytoskeleton via beta catenin, which has a pivotal role in inducing cell proliferation as mentioned above, and thus represses transcription initiation, induced by the beta catenin/LEF/TCF complex.
Biological systems include complex interactions and relationships. This complexity often disturbs concise and accurate analysis of test results. Regarding the present invention, CSCs are hard to isolate and identify. Moreover, the role of SCs in solid tumors is controversial and the understanding of universal laws concerning SCs proliferation are lacking. Thus, in silico experimentations using mathematical models may overcome these shortcomings in the study of CSCs, the factors governing their cellular fate and their involvement in tumor formation, progression and recurrence.
In [Agur Z et al, J. Math. Biol. 44, 79-86 (2002), Agur Z et al, a simple discrete model for a developing tissue was put forward and analyzed. Analysis of this model, which is represented by cellular automata on a connected, locally finite undirected graph, shows the minimal number of basic properties responsible for maintaining tissue homeostasis and guaranteeing the ability of a few stem cells to repopulate the tissue after severe perturbations. Thus it is proven theoretically that a Quorum Sensing type of control, that is, regulation of proliferation/differentiation balance by a negative feedback of SCs on their own proliferation, suffices for ensuring homeostasis both in normal and in pathological tissues. However, the molecular mechanism underlying this control still remains to be investigated.
The present invention is aimed at elucidating the molecular mechanism governing SCs and CSCs fate decisions and to identify the crucial factor, whose modulation can divert them into final differentiation. A mathematical model was constructed illustrating the major signaling pathways involved in mammary CSCs cell fate. Moreover, the mathematical model integrates also signals from the CSCs environment. Mathematical analysis of this model shows that SC niche can be formed spontaneously in the tissue, rather than requiring external delimiting structures, as is currently thought [Kirnasovsky O. U, et al Math. Model. Nat. Phenom. 3, 7, 78-89 (2008)]. Moreover, the previous assertion that homeostasis in developing tissues is mainly controlled by a negative feedback on SCs proliferation, denoted Quorum Sensing, is substantiated by this analysis, singling out the Dkk signal in the Wnt pathway as the Quorum Sensing “counter.” The analysis further suggests the relative dominance of the involved biochemical pathways in different tissue conditions.